For muscular diseases, especially degenerative diseases of skeletal muscles such as muscular dystrophy, no effective therapeutic means is available at present. If skeletal muscle cells can be prepared from patient's own bone marrow stromal cells, autologous transplantation becomes possible, which is believed to be an effective therapeutic method. Moreover, utilization of skeletal muscle cells generated from patient's own bone marrow stromal cells is expected not only in therapeutic aspects such as the aforementioned regeneration medicine, but in engineering aspects such as artificial organs with prospect of future development. Since muscle cells can be easily prepared by a culture level procedure, use of the cells is also expected in preparation of hybrid type artificial organs and the like.
As a method for inducing differentiation of bone marrow stromal cells into skeletal muscle cells, the method disclosed in Japanese Patent Unexamined Publication (KOKAI) No. 2003-144155 is known. This method comprises, as essential steps, (1) the step of isolating bone marrow stromal cells from bone marrow and culturing the cells; (2) the step of adding a demethylating agent (5-azacytidine); (3) the step of adding a cAMP-increasing agent or a cAMP analogue (forskolin), and/or a cell differentiation stimulating factor (bFGF, PDGF-AA, Heregulin); (4) the step of introducing a Notch gene and/or a Notch signaling related gene into the cells and culturing the cells; (5) the step of co-culturing said gene-introduced cells with non-introduced cells; and (6) the step of adding a cAMP-increasing agent or a cAMP analogue (forskolin).
However, the aforementioned method includes six steps, and thus has a problem that operations are very complicated. If multiple steps with complicated operations are performed, indefinite factors increases, which may lead to decrease in induction efficiency. Moreover, this method is basically proposed as a method for inducing differentiation into nerve cells, and is not optimized for selectively inducing differentiation into skeletal muscle cells. Furthermore, this method uses a mixture system containing elements other than myocytes, and therefore, the method has a problem from a viewpoint of safety for clinical application of induced skeletal muscle cells.